Electron beam apparatuses, in particular a scanning electron microscope (also referred to as SEM below) and/or a transmission electron microscope (also referred to as TEM below), are used to examine objects (also referred to as specimens) in order to obtain knowledge in respect of the properties and behaviors of the objects under certain conditions.
In an SEM, an electron beam (also referred to as primary electron beam below) is generated by means of a beam generator and focused on an object to be examined by way of a beam-guiding system. An objective lens is used for focusing purposes. The primary electron beam is guided in a grid-shaped manner over a surface of the object to be examined by way of a deflection device. Here, the electrons of the primary electron beam interact with the object to be examined. In particular interaction particles and/or interaction radiation is/are generated as a result of the interaction. By way of example, the interaction particles are electrons. In particular, electrons are emitted by the object—the so-called secondary electrons—and electrons of the primary electron beam are scattered back—the so-called backscattered electrons. The interaction particles form the so-called secondary beam and they are detected by at least one particle detector. The particle detector generates detection signals which are used to generate an image of the object. An imaging of the object to be examined is thus obtained.
By way of example, the interaction radiation is x-ray radiation or cathodoluminescence. It is detected for example with a radiation detector and is used in particular for examining the material composition of the object.
In the case of a TEM, a primary electron beam is likewise generated by means of a beam generator and focused on an object to be examined by means of a beam-guiding system. The primary electron beam passes through the object to be examined. When the primary electron beam passes through the object to be examined, the electrons of the primary electron beam interact with the material of the object to be examined. The electrons passing through the object to be examined are imaged onto a luminescent screen or onto a detector—for example in the form of a camera—by a system comprising an objective lens. By way of example, the aforementioned system additionally also comprises a projection lens. Here, imaging may also take place in the scanning mode of a TEM. As a rule, such a TEM is referred to as STEM. Additionally, provision can be made for detecting electrons scattered back at the object to be examined and/or secondary electrons emitted by the object to be examined by means of a further detector in order to image an object to be examined.
The integration of the function of an STEM and an SEM in a single particle beam apparatus is known. It is therefore possible to carry out examinations of objects with an SEM function and/or with an STEM function using this particle beam apparatus.
Furthermore, the prior art has disclosed the practice of analyzing and/or processing an object in a particle beam apparatus using, on the one hand, electrons and, on the other hand, ions. By way of example, an electron beam column having the function of an SEM is arranged at the particle beam apparatus. Additionally, an ion beam column is arranged at the particle beam apparatus. Ions used for processing an object are generated by means of an ion beam generator arranged in the ion beam column. By way of example, material of the object is ablated or material is applied onto the object during the processing. The ions are used, additionally or alternatively, for imaging. The electron beam column with the SEM function serves, in particular, for examining further the processed or unprocessed object, but also for processing the object.
The aforementioned particle beam apparatuses of the prior art each have a specimen chamber in which an object that is to be analyzed and/or processed is arranged on a specimen stage. It is furthermore known to arrange a plurality of different objects simultaneously at the specimen stage so as to analyze and/or process them one after the other using the respective particle beam apparatus that has the specimen chamber. The specimen stage is embodied to be movable so as to position the object or objects in the specimen chamber. A relative position of the object or objects with respect to an objective lens is set, for example. A known specimen stage is embodied to be movable in three directions which are arranged perpendicular to one another. Moreover, the specimen stage can be rotated about two rotational axes which are arranged perpendicular to one another.
It is known to operate the specimen chamber in different pressure ranges. For example, the specimen chamber is operated in a first pressure range or in a second pressure range. The first pressure range comprises only pressures of less than or equal to 10−3 hPa, and the second pressure range comprises only pressures of greater than 10−3 hPa. To ensure said pressure ranges, the specimen chamber is vacuum-sealed during an examination of the object or objects with the particle beam apparatus.
In order to prepare an object for an examination in a particle beam apparatus, the use of a cutting appliance in the form of a microtome is known. Accordingly, the object is prepared by cutting by means of the microtome. Therefore, the microtome is an object preparation device. The microtome has a knife with a cutting bevel. Layers of the object are cut off the object by the knife. Here, the thickness of the layers lies in the range of 5 nm to 100 μm, for example. The cut-off layers and/or an area of the object exposed by cutting is/are examined in a particle beam apparatus, for example in an SEM. Typically, biological material is prepared using the microtome. Since, as a rule, biological material has a soft embodiment, the biological material to be examined is embedded in a liquid artificial resin. The artificial resin is cured and consequently rendered cuttable. The biological material embedded in the artificial resin is introduced into the microtome. Then, layers of the biological material are ablated using the microtome and examined in the particle beam apparatus. As an alternative thereto, the exposed areas of the biological material are examined.
The practice of performing the preparation of objects by means of a microtome not only prior to introducing the objects into the specimen chamber of a particle beam apparatus but also in the specimen chamber of a particle beam apparatus itself is known. To this end, the arrangement of a microtome in the specimen chamber of a particle beam apparatus in the form of an SEM is known. A microtome that is arranged in the specimen chamber of a particle beam apparatus is also referred to as an “in situ microtome”. Using this known microtome, a layer of the object to be examined is cut, in the specimen chamber that is under vacuum, in such a way that an area to be examined is exposed. This exposed area is then examined using the particle beam of the SEM and imaged by generating an image of the exposed area. The aforementioned steps—specifically exposing an area by cutting material off the object and imaging the exposed area—can be repeated multiple times in succession in order to expose areas anew, which are then examined and imaged using the particle beam of the SEM. In this way, one image is generated in each case of each exposed area. The generated images can be used to create a 3D reconstruction of the object to be examined.
In order to obtain good imaging, the practice of aligning the areas exposed by the microtome perpendicular to the beam axis of the SEM when imaging the areas using the particle beam of the SEM is known. Moreover, the exposed areas should be positionable in the SEM in such a way that an acceptable working distance can be obtained between the objective lens of the SEM and the exposed areas. By way of example, the working distance should lie in the range of 1 mm to 5 mm. In order to obtain a perpendicular alignment of the exposed areas in relation to the beam axis of the SEM and in order to obtain a good working distance of the exposed areas from the objective lens, the practice of arranging the microtome on the adjustable specimen stage of the SEM in the specimen chamber is known. As an alternative thereto, the arrangement of a further adjustable stage for the microtome in the specimen chamber in addition to the specimen stage, the microtome being attached to said further adjustable stage, is known.
The prior art has disclosed a microtome that has a base plate and a stand arranged at the base plate. The stand is embodied as an object receptacle, at which an object to be examined is arranged. Moreover, the stand is embodied to be movable from a first position in the form of an imaging position to a second position in the form of a cutting position by way of rotation about an axis. The axis is arranged perpendicular to the optical axis of a particle beam apparatus. The known microtome has a knife that can be used to remove layers of the object and that is arranged at the cutting position of the stand. In the known microtome, the stand and consequently also the object are rotated in the direction of the cutting position by way of a rotation of the stand in a first direction (counterclockwise, for example). In the cutting position of the stand, the object strikes the knife such that a layer of the object is cut off by the knife and an area of the object is exposed. Thereupon, the stand is rotated further in the first direction in order to remove cut material that remains on the knife by way of rubbing the knife against a cleaning material. Subsequently, the stand and consequently also the object are rotated into the imaging position in a second direction (clockwise, for example). In the imaging position, the object with the exposed area is moved in the direction of the objective lens in order to set a desired working distance. As an alternative thereto, the objective lens is refocused on the exposed area. Following this, the exposed area of the object is imaged by means of the particle beam of the SEM.
As already mentioned above, it is desirable for the exposed area to be aligned perpendicular to the particle beam in order to obtain good imaging. However, on account of the arrangement of the microtome in the particle beam apparatus, it is possible that the exposed area is not aligned perpendicular to the particle beam of the particle beam apparatus. By way of example, this may be the case if the exposed area is aligned perpendicular to the vertical direction but a particle beam column of the particle beam apparatus is arranged with a tilt to the vertical.
In respect of the prior art, reference is made in an illustrative manner to WO 2015/175525 A1 and WO 2008/066846 A2.